Method of immunotherapy for treatment of human papillomavirus infection

ABSTRACT

A method of treating human papillomavirus (HPV), by administering a therapeutically effective amount of a primary cell-derived biologic to a patient infected with HPV, and inducing an immune response to HPV. A method of overcoming HPV-induced immune suppression of Langerhans cells (LC), by administering a therapeutically effective amount of a primary cell-derived biologic to a patient infected with HPV, and activating LC. A method of increasing LC migration towards lymph nodes, by administering a therapeutically effective amount of a primary cell-derived biologic to a patient infected with HPV, activating LC, and inducing LC migration towards lymph nodes. A method of generating immunity against HPV, by administering an effective amount of a primary cell derived biologic to a patient infected with HPV, generating immunity against HPV, and preventing new lesions from developing.

RELATED APPLICATIONS

This application is continuation of U.S. application Ser. No.13/514,688, filed on Jun. 8, 2012, which is a national stage filingunder 35 U.S.C. §371 of international application PCT/US2010/059450,filed Dec. 8, 2010, which claims the benefit under 35 U.S.C. §119(e) ofU.S. provisional application Ser. No. 61/267,590, filed Dec. 8, 2009,the disclosures of all of which are incorporated by reference herein intheir entireties.

TECHNICAL FIELD

The present invention relates to methods of reversing immunesuppression. In particular, the present invention relates to reversingimmune suppression in human papillomavirus (HPV) and treating HPVinfection.

BACKGROUND OF THE INVENTION

Human papillomaviruses (HPV) are a family of sexually transmitted DNAviruses with over 100 different genotypes. The genotypes are dividedinto the low-risk and high-risk categories based on the spectrum oflesions they induce. The low-risk types primarily induce benign genitalcondylomas and low-grade squamous intraepithelial lesions whereas thehigh-risk types are associated with the development of anogenitalcancers and can be detected in >99% of cervical cancers, with HPV16found in about 50% of cases. In the United States, an estimated 75% ofthe sexually active population acquires at least one genital HPV typeduring their lifetime.

While morbidity and mortality caused by cervical cancer can be reducedwith effective Papanicolaou (Pap) smear screening, early detection, andtreatment, none of these are readily available in developing countries,the origin of many immigrants to the U.S. In developing countries,cervical cancer remains the second leading cause of cancer-relateddeaths among women. Importantly, the burden of this disease is expectedto increase dramatically in the next decades due to changingdemographics. Even in the U.S. where screening programs have reduced theoverall rate of invasive cancer, a disparity exists in the incidence ofcancer development between white non-Hispanic, black, Hispanic, andeconomically disadvantaged women. Penetrance of the current FDA-approvedpreventive vaccine for HPV, GARDASIL® (Merck), in the U.S. has beendisappointing. The vaccine was administered to only 25% of girls ages13-17, 10% of all females ages 18-26, and was given to only 1.1% ofHispanic women in 2007. Moreover, the vaccine is ineffective in womenthat have already been infected with the virus, whether they havedeveloped (pre-) cancerous cervical lesions or not. Given the lifetimerisk of HPV infection and the fact that populations severelyunderrepresented in vaccine coverage will likely continue to developcervical and other HPV-related diseases at an alarming rate, it is clearthat there is an enormous need for therapeutic approaches that wouldmitigate carcinogenic effects after viral infection has occurred.

HPV are non-lytic, non-enveloped viruses. Their genome coding regionsare denoted E and L for “early” and “late” proteins. The E proteinsfulfill regulatory functions vital for genome replication, two of which(E6 and E7) play a significant role in oncogenesis, while the two Lproteins (L1 and L2) are the self-assembling capsid proteins responsiblefor DNA packaging and virion assembly. Infection by papillomaviruses isunique in that its productive lifecycle is coupled to the cellulardifferentiation of proliferating host epidermal or mucosal basalepithelial cells. HPV remains suprabasal throughout its lifecycle andtherefore only contacts cells in the epidermis such as basal cells andLangerhans cells (LC). Due to the coupling of its lifecycle to cellulardifferentiation, it is difficult to produce HPV virions in vitro. As analternative to HPV virions, HPV virus-like particles (VLP) and HPVpseudovirions, capable of carrying reporter plasmids, have beendeveloped and are both technologies that are established in thelaboratory of Applicants.

Persistence of a high-risk HPV infection is a major risk factor in thedevelopment of cervical cancer. While a majority of women infected withHPV clear the virus, the time taken to do so can range from many monthsto years. About 15% of women that have high-risk HPV infections do notinitiate an effective immune response against HPV, allowing the virus topersist for decades. The slow clearance rate and lack of effectiveimmunity indicates that HPV somehow escapes the immune response.

HPV has developed a variety of escape mechanisms that circumventimmediate elimination, allowing viral replication and persistence in thehost. Applicants have shown that HPV manipulates LC as a mechanism ofimmune escape, shown in FIG. 4. LC located in the epithelial layer ofthe skin and mucosa are the first and critical APC to come into contactwith HPV. Consequently, LC are responsible for initiating an effectiveimmune response against HPV infection. Upon recognition of a foreignantigen, LC undergo maturation, which consists of phenotypic andfunctional changes including up-regulation of co-stimulatory moleculesCD80 and CD86, MHC class I and II, chemokine receptors such as CCR7,secretion of cytokines and chemokines, and migration to regional lymphnodes. Applicants have established that LC exposed to HPV16 L1 L2 VLP donot up-regulate co-stimulatory molecules and chemokine receptors, do notsecrete cytokines and chemokines and do not initiate epitope-specificimmune responses against HPV16 VLP-derived antigens. In contrast,myeloid DC are activated by HPV16 L1 L2 VLP and once activated,stimulate HPV-specific T cells. Different intracellular signalingcascades are initiated in DC versus LC upon uptake of HPV16 L1 L2 VLP.When stimulated with HPV16 L1 L2 VLP, the mitogen-activated proteinkinase (MAPK) pathway is activated in DC whereas it is inactivated inLC. However, the phosphoinositide 3-kinase (PI3K) pathway is activatedin LC, leading to a signaling cascade that results in the inactivationof Akt. HPV16 E7-specific T cells can recognize and kill LC exposed toHPV16 L1L2-E7 chimeric VLP (cVLP), indicating that HPV peptides arepresented by LC after cVLP internalization but that HPV16 L1 L2 VLPinhibit LC from inducing an immune response. Taken together, the datasuggest that LC present HPV-derived peptides in the absence ofco-stimulation, thereby becoming tolerogenic and immune-suppressive.This in turn can lead to persistence of the HPV infection and anincreased likelihood of cancer development.

Prophylactic vaccines for HPV induce high titers of HPV-neutralizingantibodies and have shown high efficacy up to 6.5 years of follow-up andsustained levels of antibodies. However, in women infected with HPV, aphase 3 trial found no evidence of accelerated viral clearance in thevaccinated group as compared to the control group, illustratingconclusively the lack of therapeutic efficacy in preventive VLP-basedvaccines. Furthermore, with the long incubation time of HPV, severalmechanisms of immune evasion and only a few years of observation,sustained efficacy of prophylactic vaccines on cancer prevention has yetto be determined. The fact that about one third of cervical cancer iscaused by HPV types other than those currently present in the vaccinesincreases the scope of the problem. Thus, it will take decades to beable to detect a quantifiable effect on cervical cancer rates.Meanwhile, the need for therapies to treat HPV infections and associatedlesions remains for the hundreds of millions of women worldwide that arecurrently infected with high-risk HPV or will become infected in thecoming years.

Surgery, the standard of care for patients with cervical intraepithelialneoplasia (CIN) lesions, is usually quoted as being up to 90% effectivein removing CIN lesions when followed for one year. However, it is lesseffective when women are monitored over their lifetime. Greater than 80%of women that undergo surgical procedures will subsequently return inneed for a second related procedure in cases where surgery does notremove the HPV infection or where elimination of one HPV type encouragesre-activation of secondary HPV infections. The therapeutic vaccines thatare in development aim to control malignancy by activating the patient'sown cellular immune response and target antigens present in the (pre-)cancer cells. Several candidate vaccines have been developed over thelast fifteen years; however, to date, there is not a single cancervaccine that has been approved by the Food and Drug Administration.

Interventions that prevent HPV infections from reaching the stage ofinducing carcinogenesis are needed. Such interventions are feasible,since HPV infection can be detected early on with a commerciallyavailable HPV detection kit (Digene Corp.).

Several lines of evidence support the importance of the cellular immunesystem in controlling the pathogenesis of HPV and associated cervicallesions. Firstly, 25-40% of HPV positive, mildly dysplastic lesionsresolve spontaneously or shortly after local biopsy suggesting thatinduction of local inflammation may be involved with regression.Secondly, immunodeficiency is associated with increased incidence of HPVinfection. Regressing HPV-associated skin warts and genital warts oftenhave T lymphocytes in the lesions, suggesting that active cell-mediatedimmune responses to HPV may be a component of regression of the disease.Taken together, these studies illustrate that therapeutic regimens forHPV infection and its associated diseases should aim to induce strongcellular immunity at the site of infection.

Therefore, there is a need for both an effective immunological treatmentof HPV over the lifetime of women as well as a method of overcoming theHPV-induced immune suppression of LC that prevents effective treatment.

SUMMARY OF THE INVENTION

The present invention provides a method of treating human papillomavirus(HPV), including the steps of administering a therapeutically effectiveamount of a primary cell-derived biologic to a patient infected withHPV, and inducing an immune response to HPV infection.

The present invention also provides for a method of overcomingHPV-induced immune suppression of Langerhans cells (LC), including thesteps of administering a therapeutically effective amount of a primarycell-derived biologic to a patient infected with HPV, and activating LC.

The present invention provides for a method of increasing LC migrationtowards lymph nodes, including the steps of administering atherapeutically effective amount of a primary cell-derived biologic to apatient infected with HPV, activating LC, and inducing LC migrationtowards lymph nodes.

The present invention further provides for a method of generatingimmunity against HPV, including the steps of administering an effectiveamount of a primary cell derived biologic to a patient infected withHPV, generating immunity against HPV, and preventing new lesions fromdeveloping.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings, wherein:

FIG. 1 is a histogram of phenotypic markers expressed in Langerhanscells (LC) (prior art);

FIG. 2 is a graph showing CD86 expression by LC (prior art);

FIG. 3 is a diagram of unactivated LC versus activated LC;

FIG. 4 is a diagram of HPV inducing tolerization of LC;

FIG. 5 is a diagram of the experimental design of experiments on LCactivation;

FIG. 6 is a graph of upregulation of surface activation markers on humanLC exposed to HPV16 and IRX-2;

FIG. 7 is a graph showing that IRX-2 induces human LC migration in theabsence and presence of HPV16;

FIG. 8 is a graph showing that human Langerhans cells exposed to IRX-2are superior in stimulating allogeneic T cells in the presence of HPV;and

FIG. 9 is a graph showing human Langerhans cells treated with IRX-2after HPV exposure secrete high levels of IL-8 and IP-10.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides in general for methods of treating HPVand overcoming HPV-induced immune suppression of LC by theadministration of a primary cell-derived biologic (IRX-2). The treatmentof the present invention is effective in treating patients withpersistent HPV and whose immune system is not able to produce aneffective response against HPV.

As used herein, “effective amount” refers to an amount of primary cellderived biologic that is needed to achieve the desired result of thepresent invention, namely, producing a reversal of immune suppression ofLC in HPV-infected patients. One skilled in the art can determine theeffective amount of the primary cell derived biologic that should begiven to a particular patient.

“IRX-2”, also known as “citoplurikin”, is a leukocyte-derived, naturalprimary cell-derived biologic produced under cGMP standards by purifiedhuman white blood cells (mononuclear cells) stimulated byphytohemagglutinin (PHA) and ciprofloxacin (CIPRO). The major activecomponents are interleukin 1β (IL-1β, also referred to herein as IL-1),interleukin 2 (IL-2), interleukin 6 (IL-6), interleukin 8 (IL-8), tumornecrosis factor α (TNF-α), and γ-interferon (IFN-γ). Preferably, theIRX-2 used in the present invention includes these six criticalcytokines and can contain only these six critical cytokines. IRX-2 hasalso previously been referred to as an “NCM”, a natural cytokinemixture, defined and set forth in U.S. Pat. Nos. 6,977,072 and7,153,499. The terms IRX-2, primary cell-derived biologic, and NCM areused interchangeably herein.

Briefly, IRX-2 is prepared in the continuous presence of a4-aminoquinolone antibiotic and with the continuous or pulsed presenceof a mitogen, which in the preferred embodiment is PHA. Other mitogens,however, can also be used. The IRX-2 produced for administration topatients contains a concentration of IL-1β that ranges from 60-6,000pcg/mL, more preferably, from 150-1,800 pcg/mL; a concentration of IL-2that ranges from 600-60,000 pcg/mL, more preferably, from 3,000-12,000pcg/mL, and concentrations of IFN-γ and TNF-α that range from 200-20,000pcg/mL, more preferably, from 1,000-4,000 pcg/mL.

IRX-2 can also contain a concentration of IL-6 that ranges from 60-6,000pcg/mL, more preferably, from 300-2,000 pcg/mL; a concentration of IL-8that ranges from 6000-600,000 pcg/mL, more preferably from20,000-180,000 pcg/mL; a concentration of TNF-α that ranges from200-20,000 pcg/ml, more preferably, from 1,000-4,000 pcg/mL.Recombinant, natural or pegylated cytokines can be used, or IRX-2 caninclude a mixture of recombinant, natural or pegylated cytokines. IRX-2can contain only the above cytokines; however, other cytokines can beincluded. The IRX-2 of the present invention can further include otherrecombinant, natural or pegylated cytokines such as IL-7, IL-12, IL-15,GM-CSF (at a concentration that ranges from 100-10,000 pcg/mL, morepreferably from 500-2,000 pcg/mL), and G-CSF. The method of making IRX-2is disclosed in the above-cited patents as well as in U.S. patentapplication Ser. No. 12/423,601.

Also encompassed by the present invention are derivatives, fragments andpeptides related to the cytokines disclosed herein, wherein suchderivatives, fragments and peptides retain the biological activity oftheir respective cytokines.

The multiple active cytokine components of IRX-2 act on multiple celltypes of the immune system, including T cells and dendritic cells. Inclinical trials of H&NSCC patients, IRX-2 was shown to be safe,tolerable and biologically active, resulting in both apparentdisease-free survival and overall survival. The physiologic quantitiesof cytokines in IRX-2 can be administered locally, including topically,providing an opportunity to alter the microenvironment where LCencounter HPV. Natural cytokine mixtures from monocyte-conditionedmedium or mixtures of recombinant inflammatory cytokines containingTNFα, IL-1β, IL-6, and PGE2 have traditionally been used to mature DCfor ex vivo generated DC-based cancer vaccines. The physiologic cytokinelevels in IRX-2 are much lower than concentrations of recombinantcytokines used in ex vivo DC maturation or in high dose systemiccytokine therapies. The low levels of cytokines in IRX-2 allow it to beinjected directly into patients with no significant toxicity.

IRX-2 also contains several cytokines that are critical mediators of Tcell activation and proliferation. The ability of IRX-2 to activate bothDC and T cells makes it especially attractive since it is thiscombination of immune cell subsets that coordinate the immune responseagainst virus-infected cells.

Other compounds can also be administered along with IRX-2, such aschemical inhibitors, non-steroidal anti-inflammatory drugs (NSAIDS),zinc, and combinations thereof.

The chemical inhibitor can be any chemotherapeutic agent that is notimmunosuppressive (preferably used at low doses) and that hasimmunomodulatory effects so as to increase immunity and/or an immuneresponse, e.g., by inhibiting immune suppression or suppressormechanisms in the body. According to a preferred embodiment, thechemical inhibitor is an anti-neoplastic agent, including but notlimited to alkylating agents, antimetabolites and antibiotics. Thechemical inhibitor can also be an immunomodulating agent such asthalidomide. The chemical inhibitor can also be in a salt or othercomplex form. Preferably, the chemical inhibitor is the alkylating agentcyclophosphamide (CY).

The NSAID is preferably indomethacin (INDO), which is both a CoxI andCoxII inhibitor. The NSAID can also be ibuprofen or CoxII inhibitorssuch as celecoxib and rofecoxib, or combinations thereof.

The four components used together (i.e. chemical inhibitor, NSAID,primary cell derived biologic, and zinc) are able to address thesuppressive environment created by the immune target and restore thecellular immune response of the patient. More specifically, the chemicalinhibitor inhibits T regulatory cells; the NSAID reverses local immunesuppression by prostaglandins, the primary cell derived biologicactivates dendritic cells, stimulates T cells, and protects T cells fromapoptosis; and zinc provides key nutrients for T cell function. Thiscombined action encourages immune response to both endogenous andexogenous antigens.

More specifically, the present invention provides for a method oftreating HPV, by administering a therapeutically effective amount ofIRX-2 to a patient infected with HPV, and inducing an immune response toHPV. Preferably, IRX-2 includes the six critical cytokines of IL-1,IL-2, IL-6, IL-8, TNF-α, and IFN-γ as described above. Additionalcytokines can also be included as described above. Additional compoundssuch as a chemical inhibitor, NSAID, and zinc can also be administeredas described above. Preferably, the IRX-2 is administered in theepithelium by injection where HPV infection is present as well as wherethe LC are located that are needed to induce an immune response. Topicalapplication to the cervix is also preferred. Topical application methodsinclude, but are not limited to, the dispersal of IRX-2 in the liposomeformulation Biphasix™ (Helix Biopharma Corp., Aurora, Ontario, CA),followed by application to cervical epithelium; use of the Cervical DrugDelivery System™ (Cytocore®, Inc. Chicago, Ill.), wherein IRX-2 isabsorbed into a bioadhesive polymer patch, which is then affixed tocervical epithelium; and the infusion of IRX-2 solution into the cervixvia a cervical isolation and delivery apparatus such as that disclosedin U.S. Pat. No. 7,165,550 to Tracy et al.

In general, IRX-2 acts to effectively “turn on” the immune system bymaturing immature dendritic cells, stimulate the production of naïve Tcells, and effectively present antigen to the naïve T cells. IRX-2 isable to effectively treat HPV by inducing an immune response to HPV,something that is lacking in HPV patients due to the immune suppressiveactions of HPV on LC.

The immune response to HPV is induced by activating LC. Activated versusunactivated LC are shown in FIG. 3. Essentially, IRX-2 is able toovercome the immune suppression of LC by HPV. Activated LC can thensecrete T cell-activating and immune modulating cytokines in order toinduce an immune response to HPV, thus eliminating all present lesionsas well as protecting against the eruption of future lesions due to theability of the immune system to effectively attack HPV. For example,IRX-2 increases secretion of IL-8 and ILIP-10 production by LC. The LCare able to activate HPV-specific CD8+ T cells. As described in theexamples below, activation of LC is confirmed by an upregulation ofCD1a, MHC class I, MHC class II, CD40, CD80, CD83, CD86, and CCR7.

The present invention also provides for a method of overcomingHPV-induced immune suppression of LC, by administering a therapeuticallyeffective amount of IRX-2 to a patient infected with HPV, and activatingLC. The IRX-2 is described above. By activating LC, the IRX-2 is able toovercome immune suppression of LC due to HPV. Thus, the LC can noweffectively induce the activation of T cells to attack the HPV presentin the patient.

The present invention provides for a method of increasing LC migrationtowards lymph nodes, by administering a therapeutically effective amountof IRX-2, activating LC, and inducing LC migration towards lymph nodes.The IRX-2 is described above. Active LC can effectively migrate towardsa patient's lymph nodes after IRX-2 treatment and produce an immuneresponse in a patient suffering from HPV. Due to immune suppression byHPV, LC are normally not able to migrate to the lymph nodes. However,treatment with IRX-2 reverses this immune suppression so that LC areable to effectively function in the immune system.

The present invention provides for a method of generating immunityagainst HPV, by administering an effective amount of IRX-2 to a patientinfected with HPV, generating immunity against HPV, and preventing newlesions from developing. IRX-2 is able to activate LC which have beensuppressed by HPV. Activated LC can generate an immune response to HPV.Having an immune system that actively recognizes and is able to attackHPV allows for the prevention of any new lesions from developing. Anactivated immune system is able to effectively treat HPV and preventfuture development of the disease.

There are several benefits to the present invention. Since lesions areprimarily caused by persistence of HPV, interventions that induceimmunological clearance of infections and prevent the transmission ofHPV can have an enormous impact on public health. Elimination of HPVpersistence contributes to reduced health care spending for eachHPV-related cancer that would have occurred. This approach is a low-costalternative to repeated screening or expensive surgical interventionwith a high reasonable expectation of success, because it targets thecause of HPV-induced lesion development, namely HPV persistence and itsrelated immune escape.

For any of the above embodiments, the following administration detailsand/or protocols for treatment are used:

Preferably, the cytokine composition is applied locally by injection toHPV-infected epithelium. Alternatively, the cytokine composition of thepresent invention can be injected around lymphatics that drain intolymph nodes regional to a lesion or other virus infected area beingtreated. More specifically, local perilymphatic injections or otherinjections that are known to those of skill in the art are administeredto provide sufficient localization of the immunotherapy preparation.

In the embodiment wherein an exogenous antigen is to be utilized,exogenously provided synthetic or extracted antigens such as tumorantigen and peptides (see Bellone, 1998) can be administered into thepre-primed or co-primed regional or distal lymph node, either in aseparate preparation or as part of the cytokine composition of theinvention.

Endogenous suppression of T cells, which can be caused by, e.g., canceror other immunosuppressive diseases, can be blocked by theco-administration of low dose cyclophosphamide (CY) and a non-steroidalanti-inflammatory drug (NSAID) (i.e., in combination with the cytokinecompositions of the invention). The NSAID is preferably indomethacin(INDO) but ibuprofen or CoxII inhibitors such as celecoxib (CELEBREX®)or rofecoxib (VIOXX®) or combinations thereof can also be used. Sideeffects of NSAIDS can be aggressively treated with proton inhibitors andprostaglandin E analogs. Zinc and multi-vitamins, possibly including theaddition of selenium, can also be added as agents to help restore T cellimmunity. Preferably, the dose of zinc is 15 to 75 mg. A standardmultivitamin can be administered. The zinc can be an availablegluconate.

The cytokine compositions of the invention can be administered prior toor after surgery, radiotherapy, chemotherapy, or combinations thereof.The compositions of the invention can be administered during therecurrence of tumors, i.e., during a period where tumor growth isoccurring again after a period where tumors were thought to havedisappeared or were in remission.

The Mechanism of Action of IRX-2.

As defined above, the primary cell-derived biologic of the inventionacts as an adjuvant, i.e., stimulates or enhances the immune response ofa patient to a particular antigen. Moreover, the IRX-2 compositions andmethods of the invention are particularly suited to stimulate Tcell-mediated immune responses. Immune responses promoted by thecompositions and methods of the invention include the induction orgeneration of naïve T cells, the differentiation and maturation ofdendritic cells, allowing for proper presentation of antigen to T cells(e.g., in the lymph nodes), and the activation of monocytes andmacrophages. Specifically, in cancer patients, immune responses promotedby the compositions and methods of the invention include tumorinfiltration by lymphocytes, tumor fragmentation and regression as wellas a reduction in sinus histiocytosis (when present). Essentially, theprimary cell-derived biologic induces immune production and blocksimmune destruction. The mechanism of action of the primary cell-derivedbiologic is further described in U.S. patent application Ser. No.12/323,595 to Applicants.

More specifically, the compositions and methods of the present inventionaid in overcoming immune depression/suppression in patients by inducingthe production of naïve T cells. The term “naïve” T cells, as definedherein, denotes newly produced T cells, which T cells have not yet beenexposed to antigen. Thus, the compositions and methods of the inventionreplenish or generate new T cells.

Because dendritic cells are known to play such a key role in antigenpresentation in the production of an appropriate immune response invivo, an agent having a stimulatory effect on dendritic cell maturationwill act as an adjuvant in eliciting a good immune response to anantigen. The cytokine compositions of the present invention promotedendritic cell maturation. The cytokine compositions of the inventionalso provide a further adjuvant effect by acting as potent activators ofmonocytes/macrophages. Monocytes are precursors to both DCs andmacrophages in the body and thus an agent that promotesmonocyte/macrophage activation has an adjuvant effect on immuneresponses in vivo.

The primary cell-derived biologic also blocks immune destruction byprotecting the activated T cells from apoptosis. Clinical andexperimental data show that certain cytokines, especially survivalcytokines using the common receptor γ chain, are able to protectactivated T cells from tumor-induced death and enhance their anti-tumoractivity.

More specifically, there are several ways in which the primarycell-derived biologic protects T cells from apoptosis. The expression ofanti-apoptotic signaling molecules (i.e. JAK-3 and phosphor-Akt) isup-regulated and the expression of pro-apoptotic molecules (i.e. SOCS-2)is down-regulated. Activation of caspases in CD8+ and CD4+T lymphocytesis decreased and cFLIP expression is increased. Inhibition of thePI3K/Akt survival pathway is counteracted by IRX-2. The T cells areprotected from both extrinsic apoptosis (MV-induced and FasL-inducedapoptosis) and intrinsic mitochondrial apoptosis.

The protection from extrinsic MV-induced apoptosis is furtheraccomplished by preventing down-regulation of JAK3, CD3-ζ, and STATS;inhibiting dephosphorylation of Akt-1/2; and maintaining balanced ratiosof Bax/Bcl-2, Bax-Bcl-xL, and Bim/Mcl-1. The protection from MV-inducedapoptosis is also accomplished by preventing induction of the activityof caspase-3 and caspase-7. More specifically, the induction of theactive cleaved form of caspase-3 is blocked, as is the loss ofmitochondrial membrane potential. Nuclear DNA fragmentation isinhibited. Protection from intrinsic apoptosis by the primarycell-derived biologic is shown by its protection of activated T cellsfrom staurosporine-induced apoptosis.

Importantly, the cytokines of the primary cell-derived biologic protectthe activated T cells from apoptosis in a synergistic manner. In otherwords, the combination of the cytokines in the primary cell-derivedbiologic produces a greater effect than is seen by administeringindividual cytokines alone.

In view of the above, the compositions and methods of the presentinvention stimulate the immune system via multiple effects, includingthe in vivo maturation of dendritic cells resulting in effective peptideantigen presentation as well as activation of monocytes and macrophagesand the production of naïve uncommitted T cells. The proper presentationof antigen leads to T and B cell clonal expansion, creating immunity inthe patient. In the case of cancer patients, the effects noted aboveresult in the infiltration, e.g., of lymphocytes, into tumors (e.g., viahematogenous spread) and tumor reduction and/or destruction. The resultis increased survival due to immunologic memory.

The cytokine compositions of the present invention are administered anddosed to promote optimal immunization either to exogenous or endogenousantigen, taking into account the clinical condition of the individualpatient, the site and method of administration, scheduling ofadministration, patient age, sex, and body weight. The pharmaceutically“effective amount” for purposes herein is thus determined by suchconsiderations as are known in the art. The amount must be effective topromote immunization, leading to, e.g., tumor reduction, tumorfragmentation and leukocyte infiltration, delayed recurrence or improvedsurvival rate, or improvement or elimination of symptoms, includingincreased T cell counts.

In the methods of the present invention, the compositions of the presentinvention can be administered in various ways. It should be noted thatthe cytokines or exogenous antigens used in the compositions of theinvention can be administered in their standard forms or aspharmaceutically acceptable derivatives and can be administered alone oras active ingredients in combination with pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles. Furthermore, thecompositions of the invention can be administered intra- orsubcutaneously, or peri- or intralymphatically, intranodally orintrasplenically or intramuscularly, intraperitoneally, andintrathoracically. The compositions of the invention can also be appliedtopically to HPV-infected epithelium, for example by infusion into thecervix of a patient. The patient being treated is a warm-blooded animaland, in particular, mammals including man. The pharmaceuticallyacceptable carriers, diluents, adjuvants and vehicles as well as implantcarriers generally refer to inert, non-toxic solid or liquid fillers,diluents or encapsulating material not reacting with the activeingredients of the invention.

The doses can be single doses or multiple doses over a period of severaldays. When administering the compositions of the present invention, theyare generally formulated in a unit dosage injectable form (e.g.,solution, suspension, or emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, or vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, can also be used as solvent systems for the compositions ofthe invention. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it is desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent or additive usedwould have to be compatible with the cytokines or exogenous antigens ofthe invention.

Sterile injectable solutions can be prepared by incorporating thecytokines or exogenous antigens utilized in practicing the presentinvention in the required amount of the appropriate solvent with severalof the other ingredients, as desired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicles, additives, and diluents;or the cytokines and/or exogenous antigens utilized in the presentinvention can be administered parenterally to the patient in the form ofslow-release subcutaneous implants or targeted delivery systems such asmonoclonal antibodies, vectored delivery, iontophoretic, polymermatrices, liposomes, and microspheres. Examples of delivery systemsuseful in the present invention include those disclosed in U.S. Pat.Nos. 5,225,182; 5,169,383; 5,167,616; 4,959,217; 4,925,678; 4,487,603;4,486,194; 4,447,233; 4,447,224; 4,439,196; and 4,475,196. Many othersuch implants, delivery systems, and modules are well known to thoseskilled in the art.

It should be apparent that the compositions and methods of the inventionare useful for the treatment of antigen-producing diseases such ascancer, infectious diseases or persistent lesions, as discussed above.The compositions and methods promote immunization against the antigensproduced by these diseases by stimulating immune responses in patientsin vivo, which immune responses help to alleviate or eliminate thesymptoms and effects of the disease in the patient.

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided for thepurpose of illustration only, and are not intended to be limiting unlessotherwise specified. Thus, the present invention should in no way beconstrued as being limited to the following examples, but rather, beconstrued to encompass any and all variations which become evident as aresult of the teaching provided herein.

Example 1 Generation of Human LC

As HPV only infects human beings, human immune cells were used to studythe interaction of HPV with LC. Primary LC that were isolated from humanskin, becoming activated through the migration process and express highlevels of MHC and costimulatory molecules. Therefore, it is not feasibleto isolate primary unactivated skin-derived LC from human donors withwhich to conduct meaningful in vitro long term and reproduciblefunctional studies. For the experiments, primary LC were generated fromperipheral blood monocytes isolated from healthy donors usingdifferentiating cytokines ex vivo. Circulating monocytes were directprecursors of epidermal LC in vivo. Applicants and others have shownthat ex vivo derived LC express the same surface markers as epidermal LC(Langerin, E-cadherin, CD11c, CD1a, high MHC class II and intracellularBirbeck granules) (FIG. 1) and can be used consistently for in vitro LCstudies.

FIG. 1 shows that human monocyte-derived Langerhans cells expresssimilar phenotypic markers as skin-derived Langerhans cells. ImmatureLangerhans cells were differentiated from adherent monocytes in 1000IU/mL GM-CSF, 1000 IU/mL IL-4 and 10 ng/mL TGFβ for 7 days. Cells wereanalyzed by flow cytometry for the expression of MHC class II(HLA-DP,DQ,DR), CD1a, CD11c, Langerin, or E-cadherin (gray shadedhistograms). Isotype controls are shown as black unshaded histograms.Data are representative of LC derived from several healthy donors.

Reversal of HPV Immune Escape

Applicants have previously embarked on strategies to reverse HPV immuneescape by targeting LC. LC express a variety of TLRs, including TLR3, 7,8, and 9, which recognize pathogen-associated molecular patterns (PAMPs)and upon engaging their ligands activate the cell. Surprisingly, it wasfound that a TLR 7 agonist ALDARA® (Imiquimod, Graceway Pharmaceuticals,LLC), FDA-approved for external genital warts, had absolutely no effecton LC activation, as measured by expression of CD86 (FIG. 2). This mightexplain the yet unpublished observations that the use of Imiquimod fortreating cervical lesions is not effective. Interestingly, a TLR 8activator (3M-002) and a TLR7/8 agonist (Resiquimod) fully activated HPVinfected LC such that they started to induce HPV-specific T cellresponses in vitro. These data indicate that suppression of LC functionby HPV can be reversed by activating certain “danger signal” pathways.

Activation of Human Langerhans Cells with IRX-2

The ability of LC to efficiently stimulate T cells after migration fromthe epidermis to the draining lymph nodes after exposure to pathogens orother “danger signals” requires the expression of costimulatorymolecules and chemokine receptors on their cell surface. The effect ofIRX-2 on the phenotypic maturation of human monocyte-derived LC in vitrowas examined. IRX-2 treatment was found to induce upregulation of bothMHC class I and MHC class II, and the costimulatory molecules CD40, CD80and CD86, the maturation marker CD83, and LC activation was performed aspreviously described. Briefly, LC were harvested, washed, and eitherleft untreated or treated with HPV16L1L2 VLP at a concentration of 10μg/106 cells for 1 hour at 37° C. Following the incubation, the cellswere placed at 37° C. for 6 hours in complete medium. Next, the cellswere left untreated or treated with IRX-2 (1:2 dilution) or with 1 μg/mLLPS as a positive control. The cells were incubated for an additional 48hours post IRX-2 treatment. Cells were harvested, washed, and stainedfor flow cytometric analysis staining for CD1a, MHC class I, MHC classII, CD40, CD80, CD83, CD86, or isotype controls. The fold change ofsurface markers between treatment groups were calculated from MFIvalues. Increased MFI indicates upregulation of markers and activationof LC. Data are representative of three individual donors

The source of IRX-2 was supernatant collected from human peripheralblood mononuclear cells stimulated by phytohemagglutinin (PHA) for a24-hour period. QC testing was used to test levels of cytokines in themixture and standardize according to the levels of four key cytokines.The cGMP manufacturing process yielded a highly consistent product withvery similar cytokine concentrations from lot to lot. To ensureconsistency in LC activation by IRX-2, two different lots of IRX-2 wereused to activate LC.

As shown in FIG. 6, expression of all six LC markers was stronglyelevated by IRX_2 treatment, regardless of whether the LC had beenexposed to HPV16 L1 L2 VLP. Interestingly, elevation of thecostimulatory molecule CD86 was even more effective in HPV16 LIL2 VLPexposed LC than in controls. Greater than 95% of cells expressed CD86and greater than 80% of LC expressed the maturation marker CD83 (notshown).

The results indicate that that IRX-2 is a potent inducer of LCactivation and maturation, and that its effectiveness is not impaired byprior exposure of LC to HPV.

Example 2

Establishing whether IRX-2, which is currently available for clinicaluse, can activate LC previously exposed to HPV16 is necessary. Theeffect of IRX-2 on LC expression of activation markers was reported inExample 1, In the next experiments, the strength of IRX-2 was tested bymeasuring cytokine secretion, migration, and activation ofalloantigen-specific T cells.

Activation of APC, like LC, is required for successful interaction withand activation of primary T lymphocytes. Inflammatory cytokines have theability to activate APC, resulting in maturation and an increase inantigen-presenting function. IRX-2 is a promising immune modulator thathas the potential to influence the immunostimulatory capacity of LC whenapplied locally to HPV-infected epithelium. In order to determinewhether IRX-2 phenotypically and functionally activates LC exposed toHPV, cytokine/chemokine secretion and migration as well as the abilityto stimulate alloantigen-specific T cell responses was performed. Thesestudies defined IRX-2 as able to reverse the immune suppression by HPV,similar to TLR8 agonists.

Experimental Design:

LC expression levels of T cell co-stimulatory markers was determinedwhen LC were exposed to HPV16 and then to IRX-2. LC were differentiatedfrom peripheral blood monocytes isolated from healthy donors. LC wereexposed to HPV16 virus-like particles (VLP) for 6 hours, and then IRX-2added for an additional 48 hours, as shown in FIG. 5. Cell surfacemolecules were measured by flow cytometry using fluorescent antibodies.Cell culture supernatants were tested for the presence ofimmune-stimulatory cytokines and chemokines to determine whether LC havebecome activated and are secreting T cell-activating or other immunemodulating cytokines. LC migration after exposure to HPV VLP and IRX-2was measured by in vitro migration through a transwell membrane. Thecapacity to stimulate T cells was evaluated by culturing LC withallogeneic T cells in a mixed lymphocyte reaction (MLR). Untreated LC,LC exposed to HPV VLP alone, and LC treated with IRX-2 alone serve ascontrols in each experiment. Each experiment was repeated at least 3times using LC derived from individual healthy donors. Subjects werechosen who are HLA-A*0201 positive, so that well defined T cell immuneresponses were measured against known HPV-derived peptide antigens.These data show that IRX-2 enables HPV-exposed LC to gain back theircapacity to stimulate T cells and to migrate—both functions needed for aproductive anti-viral immune response.

Generation of Human Langerhans Cells:

Primary LC were generated from commercially available peripheral bloodmonocytes isolated by leukapheresis of anonymous healthy donors usingdifferentiating cytokines ex vivo. Monocyte-derived LC were generatedfirst through plastic adherence of cryopreserved PBMC to culture flasks.Adherent cells were cultured for 7 days in medium containing 1000 U/mLrecombinant human (rhu)-GM-CSF, 1000 U/mL rhu-IL-4 and 10 ng/mLrhu-TGF-β1, replenished twice during the culture period.

LC Migration:

Chemokine directed migration of LC was carried out using 24-wellTranswell plates with 5 μm-pore-size polycarbonate filters (CorningCostar). Media was added to the lower chamber containing either 250ng/ml rhu CCL21 (R&D Systems) or medium alone to control for spontaneousmigration. LC untreated or treated as described above, were added to theupper chamber and incubated for 4 hours at 37° C. The cells thatmigrated to the lower chamber were counted using a hemacytometer orautomated cell counter, and CCL21-dependent migration was calculated asthe ratio of cells that migrated with CCL21 to cells that migratedwithout CCL21, termed migration index. Increased migration indexindicated a functional activation of LC to move from the tissue towardsdraining lymph nodes (FIG. 7).

IRX-2 treatment produced a three to four fold increase in migration inboth control and HPV L1 L2 VLP. The results indicate that IRX-2 canpromote the migration of LC to regional lymph nodes, even when the LChad been exposed to HPV.

Mixed Lymphocyte Reaction Assay:

LC were treated with HPV16 L1 L2 VLP and IRX-2 as described above. LCwere co-cultured with untouched allogeneic T cells isolated using a MACSnegative selection human pan T cell isolation kit. Responder T cells andstimulator LC were cultured at R:S ratios of 10:1 and 5:1 for 5 days. Tcells cultured alone, LC cultured alone, T cells cultured withautologous PBMC and T cells cultured with the T cell mitogen PHA servedas controls for the assay. Radioactive ³H-thymidine-pulsed cells wereharvested and radioactivity counted on a scintillation plate counter.Radioactive cpm were determined and compared between treatments.Increased thymidine incorporation was indicative of greater T cellproliferation. Increased T cell proliferation after HPV16 L1L2 VLPexposure and IRX-2 stimulation confirmed that HPV16 L1L2 VLP-exposed LCwere gaining back their immunostimulatory capacity in the presence ofHPV (FIG. 8).

Cytokine and Chemokine Analysis:

Supernatants were collected from LC stimulated with IRX-2 and tested forsecreted cytokine and chemokines. LC were treated as described above. LCwere washed 36 hours post IRX-2 treatment and cultured for an additional36 hours, prior to collection of supernatants. This eliminatedmeasurement of cytokines present in IRX-2 mixture. The assays werecompleted using the Bio-Plex Suspension Array System which allowed forseveral inflammation and T cell stimulating and chemoattracting analytesto be assayed at once. The assayed cytokines and chemokines includedIL-8, IFN-γ Inducible Protein 10 (IP-10), Monocyte ChemoattractantProtein (MCP)-1, Macrophage Inflammatory Protein (MIP)-1α, MIP-1β, andRANTES. Data were analyzed by comparing the cytokine and chemokineconcentrations in the supernatants of treatment groups. IncreasedTh1-associated cytokine and chemokine secretion indicates functionalactivation of LC that would support the induction of CD8+ T cellresponses, while increased suppressive cytokines suggests a tolerizingor suppressive function of LC.

Treatment of LC with IRX-2 significantly increased the secretion of twoTh1 associated chemokines, IP-10 and IP10 (FIG. 9). Both chemokines arepro-inflammatory, and IP-10 is known to attract T cells, NK cells, andmonocytes to sites of inflammation. The results show that, under IRX-2stimulation, L1 L2 VLP-exposed LC were gaining back theirimmunostimulatory capacity in the presence of HPV.

CONCLUSION

IRX-2 phenotypically and functionally activated human LC from healthydonors, even after they were exposed to HPV L1 L2 VLP. Increased levelsof MHC and surface activation molecules, increased secretion ofinflammatory and T cell activating cytokines and chemokines, and anincreased ability to perform chemokine-directed migration after HPV andIRX-2 treatment (FIGS. 6-9), These results all support the effectivenessof the present invention in overcoming HPV-induced tolerization of LCand providing treatment for HPV infections. The invention has beendescribed in an illustrative manner, and it is to be understood that theterminology which has been used is intended to be in the nature of wordsof description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

1.-23. (canceled)
 24. A method of immunotherapy for treating a viralinfection caused by human papillomavirus (HPV) in a patient, comprising:administering an effective amount of a primary cell-derived biologic toa patient having a viral infection caused by HPV in order to alleviateor eliminate the infection, such that an immune response is induced tothe HPV in the patient which does alleviate or eliminate the infection,and wherein the primary cell-derived biologic includes the cytokinesinterleukin (IL)-1, interleukin (IL)-2, interleukin (IL)-6, interleukin(IL)-8, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha.25. The method of claim 24, wherein the primary cell-derived biologicincludes a concentration of IL-1 from 60-6,000 pcg/mL, a concentrationof IL-2 from 600-60,000 pcg/mL, a concentration of IL-6 from 60-6,000pcg/mL, a concentration of IL-8 from 6000-600,000 pcg/mL, aconcentration of TNF-alpha from 200-20,000 pcg/mL, and a concentrationof IFN-gamma from 200-20,000 pcg/mL.
 26. The method of claim 24, whereinthe primary cell-derived biologic further includes interleukin (IL)-7,interleukin (IL)-12, interleukin (IL)-15, granulocyte-macrophagecolony-stimulating factor (GM-CSF), and granulocyte colony-stimulatingfactor (G-CSF).
 27. The method of claim 26, wherein the primarycell-derived biologic includes a concentration of IL-7 from 100-10,000pcg/mL, a concentration of IL-12 from 100-10,000 pcg/mL, a concentrationof IL-15 from 100-10,000 pcg/mL, a concentration of GM-CSF from100-10,000 pcg/mL, and a concentration of G-CSF from 100-10,000 pcg/mL.28. The method of claim 24, wherein the method further comprises:administering an effective amount of one or more of a chemicalinhibitor, a non-steroidal anti-inflammatory drug (NSAID), and zinc tothe patient.
 29. The method of claim 28, wherein the chemical inhibitoris cyclophosphamide.
 30. The method of claim 28, wherein the NSAID isindomethacin.
 31. The method of claim 24, wherein the primarycell-derived biologic is in a sterile, injectable solution.
 32. Themethod of claim 24, wherein the administering of the primarycell-derived biologic is injection into the patient's epithelium. 33.The method of claim 24, wherein the administering of the primarycell-derived biologic is topical application to the patient'sepithelium.
 34. The method of claim 24, wherein administration of theprimary cell-derived biologic prevents new HPV-induced lesions fromdeveloping.
 35. The method of claim 24, wherein the patient has notreceived surgery, radiotherapy, chemotherapy, or combinations thereof,prior to administration of the primary cell-derived biologic.
 36. Themethod of claim 24, wherein the patient is a man.
 37. The method ofclaim 36, wherein the patient does not have cancer.
 38. The method ofclaim 24, wherein the patient is a woman.
 39. The method of claim 38,wherein the patient does not have cancer.